Effects of methionine supplementation in a reduced protein diet on growth performance, oxidative status, intestinal health, oocyst shedding, and methionine and folate metabolism in broilers under Eimeria challenge

Background This study investigated effects of different methionine (Met) supplementation levels in a reduced protein diet on growth performance, intestinal health, and different physiological parameters in broilers under Eimeria challenge. A total of 600 fourteen-day-old Cobb500 male broilers were challenged with E. maxima, E. tenella, and E. acervulina, and randomly allocated in a 2 × 5 factorial arrangement. Birds received normal protein diets (20% crude protein, NCP) or reduced protein diets (17% crude protein, LCP), containing 2.8, 4.4, 6.0, 7.6, and 9.2 g/kg of Met. Results On 6 and 9 days post inoculation (DPI), increasing Met level linearly improved the growth performance (P < 0.05). Total oocyst shedding linearly increased as Met level increased (P < 0.05). Duodenal villus height (VH):crypt depth (CD) in the LCP groups were higher on 6 DPI (P < 0.01) while lower on 9 DPI (P < 0.05) compared to the NCP groups. Jejunal CD and duodenal VH:CD changed quadratically as Met level increased (P < 0.05). On 6 DPI, liver glutathione (GSH) and glutathione disulfide (GSSG) linearly increased as Met level increased (P < 0.05). On 9 DPI, GSSG quadratically increased, whereas GSH:GSSG quadratically decreased as Met levels increased (P < 0.05). The expression of amino acid transporters linearly decreased as Met level increased (P < 0.05). The expression of zonula occludens 2 and claudin-1 linearly increased on 6 DPI whereas decreased on 9 DPI as Met level increased (P < 0.05). The expressions of cytokines were lower in the LCP groups than the NCP groups (P < 0.05). Interaction effects were found for the expression of IL-10 and TNFα on 6 DPI (P < 0.05), where it only changed quadratically in the NCP group as Met level increased. The expression of Met and folate metabolism genes were lower in the LCP groups than the NCP groups on 9 DPI (P < 0.05). The expression of these genes linearly or quadratically decreased as Met level increased (P < 0.05). Conclusion These results revealed the regulatory roles of Met in different physiological parameters including oxidative status, intestinal health, and nutrient metabolism in birds fed reduced protein diet and challenged with Eimeria.


Background
Coccidiosis is a widespread and economically significant parasitic disease affecting the poultry industry [1].It is caused by the protozoan parasite of the genus Eimeria, which inflicts damage to the intestinal lining of the birds during its reproduction cycle [2].The infection leads to compromised intestinal integrity, hindered nutrient absorption, inflammation, and oxidative stress, ultimately resulting in a substantial decline in growth performance and even mortality [3][4][5]6].Anticoccidial drugs have traditionally served as an effective method to combat coccidiosis [2].However, concerns have been raised due to the development of drug resistance [7] and growing public apprehension regarding antibiotic use in animal production [8].While vaccinating birds against coccidiosis has demonstrated effectiveness, achieving successful vaccination requires exposing birds to live or attenuated oocysts to develop immunity [9], which can still lead to intestinal lesions that might potentially predispose another intestinal disease necrotic enteritis [10].With both approaches facing limitations, the exploration of alternative strategies to mitigate the impact of coccidiosis has become necessary.
Since optimal nutrient compositions in diets are crucial for sustaining the well-being of infected birds and the altered physiological state and heightened immune responses induced by coccidiosis may lead to changes in the nutrient requirements which were proposed for the healthy broilers [11,12].One promising alternative avenue to combat coccidiosis emerged as the nutritional interventions by increasing the supplementation of nutrients possessing function roles like amino acids into broiler diets, aiming to reduce intestinal damage and promote recovery [13].Methionine (Met) as one of the essential amino acids is also considered the first limiting amino acid in poultry production [14,15].It not only plays a crucial role in maintaining bird growth and protein synthesis, but also holds significant importance in supporting the intestinal health, immune responses, and anti-oxidative functions of the birds [16][17][18][19][20][21].Besides the established significance in protein synthesis, Met also plays a crucial role in regulating several signaling pathways, including the mTOR and Wnt/β-catenin pathways [22,23].These pathways are known for their role in maintaining intestinal structure by regulating the renewal and differentiation of intestinal stem cells [24].This dual role of Met may contribute significantly to the regenerative capacity of the intestine, especially during coccidiosis infection when the intestinal epithelial cells as well as the tight junctions between them are severely damaged [5,6].Moreover, research has shown that Met possesses potent antioxidant capacity, attributed to its close metabolic relationship with glutathione (GSH) and its capability to scavenge free radicals [25][26][27][28][29]. Additionally, the critical role Met plays in the immune responses cannot be ignored, research showed that the Met as well as its metabolites S-adenosylmethionine (SAM) are important for the activation and proliferation of T cells [30,31], which are essential in combating the Eimeria infections [32].Sufficient supplementation of Met has also been shown to enhance antibody production in broilers [33][34][35].Given the multiple functional roles Met possesses, supplementation of Met in diets of broilers under Eimeria challenge might potentially alleviate the impact of the infection and improve the performance of the birds.
However, the potential negative impacts of excessive Met supplementation should not be overlooked.Toxicity caused by excessive Met supplementation has been well documented [36].While the demands for Met may increase in birds challenged by Eimeria due to its beneficial functional roles, over supplementation of Met could still lead to impaired growth performance.This negative effects could be intensified when the birds are under coccidiosis infection.Moreover, it's crucial to acknowledge the intimate connection between Met and folate metabolism within the one-carbon cycle.In the process of homocysteine remethylation, elevated Met levels might contribute to the synthesis of additional tetrahydrofolate, ultimately converted into pyrimidine for DNA synthesis and cellular proliferation [37,38].This metabolic interplay suggests that an excess of Met might potentially heighten folate and pyrimidine availability to Eimeria, which are essential for their reproduction [39,40], thus potentially exacerbating the severity of the infection by favoring the reproduction of the parasites.
Reducing the dietary protein levels in animal diets has been proposed by researchers and producers due to its various benefits [41].One major advantage is the potential to lower production costs by decreasing the usage of high-quality protein ingredients.[13,42].Additionally, this approach offers environmental advantages by decreasing nitrogen excretion and ammonia emissions [41][42][43].However, with the protein content reduced in the diets, it is of more importance to meet the optimal amino acid requirements to maintain the growth performance of the birds especially when the birds are under disease conditions [44,45].Hence, an understanding of the optimal Met supplementation levels becomes crucial for coccidia-infected birds fed a reduced protein diet, which ensures not only the economic efficiency and environmental sustainability of poultry production but also the wellbeing of the birds in the face of coccidiosis challenges.Despite the significance of this issue, there is a scarcity of studies addressing this topic.Therefore, the present study aims to bridge this gap by investigating the effects of different levels of Met supplementation in a reduced protein diet on the growth performance, intestinal health, immune responses, oocyst shedding, and metabolism of Met and folate in broilers challenged with Eimeria.Our hypothesis is that increasing Met levels could enhance the performance of broilers under Eimeria challenge by improving the oxidative status and intestinal health of the birds.However, we postulate that an optimal level of dietary Met may exist, beyond which further supplementation could potentially favor the reproduction of parasites and result in adverse effects.

Materials and methods
All the animal experiment procedures used in this study were approved by the Institutional Animal Care and Use Committee of the University of Georgia (A2021 12-012).

Birds, diets, and Eimeria challenge
A total of 600 one-day-old Cobb500 male broiler chicks were fed a same starter diet that met the breeder's nutrient recommendations [46]

Growth performance and sample collection
Body weight (BW) was measured on 0, 6, and 9 days post inoculation (DPI) for the calculation of body weight gain (BWG).Feed intake (FI) was measured daily after inoculation.Feed conversion ratio (FCR) was calculated from BWG and FI.Mortality was monitored and recorded daily.On 6 and 9 DPI, one bird per cage was randomly selected and euthanized for sample collections.Samples from liver, jejunal mucosa, and cecal tonsils (CT) were collected and snap-frozen in liquid nitrogen.The samples were stored at -80 °C for oxidative status and gene expression analyses.Approximately 2 cm in length of the duodenum, jejunum, and ileum samples were collected, rinsed with PBS, and fixed in 10% formalin for intestinal morphology analysis.Excreta were collected daily from 1 to 9 DPI from each cage for the measurement of oocyst shedding.

Intestinal morphology and intestinal permeability
The intestinal samples were removed from the 10% formalin after fixation and subsequently embedded in paraffin blocks.The sample blocks were sliced into 4 μm sections and stained with hematoxylin and eosin.
The image of the stained tissues was observed and captured under a light microscope with 2 × magnification (BZ-X800, Keyence Inc., Itasca, IL, USA).The villus height (VH) and crypt depth (CD) were measured and VH:CD ratio were calculated as described previously [4].On 5 DPI, one bird per cage was gavaged with 1 mL of fluorescein isothiocyanate dextran (FITC-d; 2.2 mg/mL, MW 4000; Sigma-Aldrich, St. Louis, MO, USA) to measure intestinal permeability.Two hours after the gavage, the birds were euthanized for blood collection.The blood was centrifuged at 1,000 × g for 15 min (Eppendorf Centrifuge 5430R, Eppendorf, Hamburg, Germany), and 100 μL of serum was used to determine the FITC-d concentration according to the method described previously [48].

Oocyst shedding
The oocyst shedding was measured according to a previously reported method [49].Briefly, 5 g of the collected excreta samples were weighed and combined with 25 mL of water.The mixture was vigorously vortexed, and 1 mL of the diluted samples was then mixed with 9 mL of saturated salt solution and vortexed thoroughly.The prepared samples were loaded into a McMaster chamber (Jorgensen Laboratories, Loveland, CO, USA) and examined under a microscope (FEC Source, Grand Ronde, OR, USA).The oocysts shedding of E. acervulina, E. maxima, and E. tenella were distinguished by their distinct oocyst sizes and shapes [50].The oocysts were quantified, and the results were expressed as the log 10 of oocysts per gram of excreta (OPG).

Oxidative status analyses
Concentrations of malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), and activities of glutathione peroxidase (GPX) and superoxide dismutase (SOD) in the liver were determined using commercial assay kits (GSH, GPX, SOD assay kits, Cayman chemical, Ann Arbor, MI, USA; MDA, BioAssay Systems, Hayward, CA, USA).Protein concentrations of the liver samples were measured by bicinchoninic acid assay (BCA) kit (Thermo Scientific, Rockford, IL, USA) to standardize the results obtained as described previously [51].

Reverse transcription and real-time PCR analysis
Liver, jejunal mucosa, and CT samples were homogenized with a MiniBeadBeater-16 (BioSpec Products Inc., Bartlesville, OK, USA), and RNA was extracted using QIAzol Lysis Reagents (Qiagen, Germantown, MD, USA) following the manufacturer's instructions.

Growth performance
The daily FI began to decline from 4 DPI, reaching its lowest point on 6 DPI.Subsequently, it gradually increased from 6 to 9 DPI (Fig. 1A).Significant interaction effects were observed for the daily FI of 1-3 DPI (P < 0.05) (Table 3).Specifically, the daily FI linearly or quadratically increased as Met levels increased in the NCP groups (P < 0.01), whereas they were not affected by Met levels in the LCP groups (Fig. 1B-D).A significant interaction effect was observed for the daily FI of 4 DPI (P < 0.01).Specifically, as Met level increased, daily FI changed quadratically in the NCP groups (P < 0.05), whereas it linearly decreased in the LCP groups (P < 0.01) (Fig. 1E).On 5 DPI, daily FI linearly decreased as Met levels increased in the LCP groups (P < 0.05) (Fig. 1F).
The daily FI of 6 DPI linearly decreased as Met levels increased (P < 0.01) (Fig. 1G) and it was significantly higher in the Met 2.8 groups than the Met 6.0, Met 7.6, and Met 9.2 groups (P < 0.01).On 6 DPI, BW and BWG linearly increased, and FCR linearly decreased as Met level increased (P < 0.05) (Fig. 2A-C).An interaction effect was observed for the FI of 0-6 DPI (P < 0.05) where it linearly decreased as Met level increased in the LCP groups while not in the NCP groups (Fig. 2D).The FCR of 0-6 DPI was significantly lower in the Met 6.0 groups compared to the Met 2.8 groups (P = 0.019) (Table 4).The FCR of 7-9 DPI quadratically decreased as Met level increased (P < 0.05) (Fig. 2E), and it was significantly lower in the Met 6.0 groups compared to the Met 2.8 groups (P = 0.018).The FCR of 0-9 DPI linearly decreased as Met level increased (P < 0.05) (Fig. 2F).No treatment effects were observed for the mortality on any timepoints.

Intestinal morphology
On 6 DPI, the duodenal CD was lower in the LCP groups compared to the NCP groups (P < 0.01) (Table 5).The duodenal VH:CD ratio was higher in the LCP groups than in the NCP groups (P < 0.01).On 6 DPI, the jejunal Table 7 Effects of dietary methionine levels and protein contents on expression of tight junction proteins and amino acid transporters of broilers challenged with Eimeria spp. on 6 day post inoculation a,b Means within a column lacking a common superscript differ (P < 0.05) CD changed quadratically as it decreased initially and then increased as Met level increased (P < 0.05) (Fig. 3A).On 9 DPI, the duodenal VH:CD ratio was lower in the LCP groups than in the NCP groups (P < 0.05) (Table 6).An interaction effect was observed for the duodenal VH (P < 0.05), where the LCP diet significantly decreased VH only in the Met 6.0 group.(Fig. 3B).The VH:CD ratio changed quadratically as it decreased initially and then increased as Met level increased (P < 0.05) (Fig. 3C).No significant effects on the ileal morphology were observed on 6 and 9 DPI.

Gene expression of tight junction proteins
On 6 DPI, the expression of claudin-1 (CLDN1) was higher in the LCP groups than in the NCP groups (P < 0.01) (Table 7).The expression of CLDN1 and zonula occludens (ZO2) linearly increased as Met level increased (P < 0.05) (Fig. 4A and B).On 9 DPI, an interaction effect was observed for the expression of CLDN1( P < 0.05), where it linearly decreased as Met level increased in the NCP groups while not in the LCP groups (Fig. 4C).The expression of ZO2 quadratically decreased as Met level increased (P < 0.05) in the NCP groups (Fig. 4D).No significant CP or Met main effects were observed for the expression of tight junction protein on 9 DPI (Table 8).

Gene expression of amino acid transporters
On 6 DPI, the expression of b(0, +)-type amino acid transporter 1 (SLC7A9) and L-type amino acid transporter 4 (SLC43A2) was lower in the LCP groups than in the NCP groups (P < 0.05) (Table 7).The expression of sodium-dependent neutral amino acid transporter B(0) AT1 (SLC6A19) and SLC7A9 linearly decreased as Met level increased (P < 0.05) (Fig. 5A and B).On 9 DPI, the expression of SLC43A2 in the LCP groups and SLC6A19 linearly decreased as Met level increased (P < 0.05) (Fig. 5C and D).No significant effects were observed for the expression of amino acid transporters on 9 DPI (Table 8).

Gene expression of cytokines
On 6 DPI, the expression of interleukin-1β (IL1β) was higher in the Met 4.4 group than in the Met 2.8 and Met 6.0 groups (P < 0.01) (Table 9).The expression of transforming growth factor β (TGFβ) was higher in the NCP groups than in the LCP groups (P < 0.05).Significant interaction effects were observed for the expression of interleukin-10 (IL10) and tumor necrosis factor α (TNFα) where their expression exhibited a quadratic trend, decreasing initially and then increasing as Met level increased in the NCP groups while not in the LCP groups (Fig. 6A and B).The expression of TGFβ exhibited a quadratic trend (P < 0.05), decreasing initially and then increasing as Met level increased in the NCP groups (Fig. 6C).On 9 DPI, the expression of IL10 was higher in the NCP groups than the LCP groups (P < 0.05) (Table 9).An interaction effect was observed for the expression of TNFα (P < 0.05), where LCP diet decreased its expression only in the Met 6.0 and Met 9.2 groups (Fig. 6D).
Liver GSSG quadratically increased as Met level increased (P < 0.01) (Fig. 7B).An interaction effect was observed for the GSH:GSSG ratio (P < 0.01), where the ratio quadratically decreased as Met level increased in the NCP groups, whereas the ratio exhibited a quadratic trend, decreasing initially and then increasing as Met level increased in the LCP groups (Fig. 7C).On 9 DPI, liver GSSG were significantly lower in the Met 2.8 groups compared to the other groups (P < 0.01) (Table 10).The GSH:GSSG ratio was significantly lower in the Met 4.4 groups compared to the Met 2.8 groups (P < 0.05).The MDA concentration and SOD activity linearly increased as Met level increased in the LCP groups (P < 0.05) (Fig. 8A and B).An interaction effect was observed for the GSH (P = 0.01), where GSH linearly increased as Met level increased in the NCP groups, whereas GSH exhibited a quadratic trend, increasing initially and then decreasing as Met level increased in the LCP groups (Fig. 8C).Liver GSSG quadratically increased whereas GSH:GSSG ratio quadratically decreased as Met level increased (P < 0.05) (Fig. 8D and E).

Oocyst shedding
The oocysts of E. acervulina were first detected in the excreta on 3 DPI, and the oocysts of E. maxima and E. tenella first appeared in the excreta on 5 DPI.The OPG of three species peaked at 6 DPI (Fig. 9A).The accumulated OPG from 1 to 9 DPI of E. tenella was significantly lower in the Met 4.4 and Met 6.0 groups than the Met 9.2 groups (Table 11).The accumulated OPG from 1 to 9 DPI of E. acervulina and E. maxima linearly increased as Met level increased (P < 0.05) (Fig. 9B and 9C).The accumulated OPG from 1 to 9 DPI of E. tenella changed quadratically as Met level increased (P < 0.05) (Fig. 9D).

Gene expression of methionine and folate metabolism enzymes
No significant effects were observed for expression of Met and folate metabolism enzymes on 6 DPI (Table 12).On 9 DPI, the expression of methionine adenosyltransferase 1A (MAT1A), methionine synthase (MTR), and cystathionine beta synthase (CBS) were lower in the LCP groups than in the NCP groups (P < 0.05).The expression of MAT1A was significantly higher in the Met 2.8 groups than the other groups (P < 0.01).The expression of adenosylhomocysteinase like 1 (AHCYL1) was higher in the Met 2.8 groups than the Met 6.0 and Met 9.2 groups (P < 0.05).The expression of CBS and AHCYL1 linearly decreased and the expression of MAT1A and MTR quadratically decreased as Met level decreased (P < 0.05) (Fig. 10A-D).On 6 DPI, the expression of thymidylate synthase (TYMS) in the LCP groups linearly increased as Met level increased (P < 0.05) (Fig. 11A).On 9 DPI, the expression of dihydrofolate reductase (DHFR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) were lower in the LCP groups than in the NCP groups (P < 0.01) (Table 13).The expression of TYMS was significantly higher in the Met 2.8 groups than the other groups (P < 0.01).The expression of methylenetetrahydrofolate reductase (MTHFR) in the LCP groups and TYMS linearly decreased as Met level increased (P < 0.05) Fig. 11B and C.

Table 9 Effects of dietary methionine levels and protein contents on expression of cytokines of broilers challenged with Eimeria spp.
a−c Means within a column lacking a common superscript differ (P < 0.05)

Discussion
The present study investigated the impact of dietary Met levels and protein content on growth performance, intestinal health, oxidative status, and gene expression related to methionine and folate metabolism enzymes in broiler chickens.The multifaceted evaluation of these parameters provided a comprehensive understanding of how Met levels and low dietary protein affect the performance and health of broilers under Eimeria challenge.
The daily FI began to decrease on 4 DPI and reached its lowest point on 6 DPI.This decrease in daily FI in response to Eimeria challenge aligned with the reproductive cycle of the parasites.It has been shown that Eimeria spp.typically undergo the asexual reproduction (schizogony) to produce merozoites from 3 to 5 DPI, the merozoites subsequently re-enter the enterocytes to initiate sexual reproduction (gametogony) around 6 to 7 DPI, resulting in severe damage to the intestinal mucosa [6,53].This damage to the intestinal mucosa and integrity may cause pain and discomfort in the birds, potentially leading to anorexia [54].After 6 DPI, the birds gradually recovered from the infection, and the FI increased accordingly.The same pattern has been reported by previous studies wherein broilers were also subjected to Eimeria challenge, and the daily change in FI was monitored [3,4,6,10].
In the present study, we observed that in the first three DPI, the FI linearly or quadratically increased as Met levels increased from deficiency to levels higher than recommendation in the NCP groups.According to studies conducted by previous researchers who fed broilers with diets containing graded levels of Met, Met deficiency could lead to suppression in FI, which be restored by increasing Met inclusion in diet [21,55,56], as observed in this current study in the NCP groups.However, the birds did not respond significantly to the changes of dietary Met levels in the LCP groups, this observation suggested that the birds' response to dietary Met levels might be dampened by the decreased CP content in the diet.To the authors' knowledge, this discrepancy in the response of FI to dietary Met levels across different levels of CP diet has not been previously reported.Further studies are warranted to elucidate the underlying mechanism behind this difference.More intriguingly, starting from 4 DPI corresponding with the reproduction of Eimeria spp.and the subsequent drop in FI, birds in the higher Met groups began to exhibit lower FI, especially in the LCP groups.By 6 DPI, when the infection was most acute, FI linearly decreased as Met levels increased.This observation suggested that during the acute infection of coccidiosis, higher dietary Met levels might exert adverse effects on the birds, potentially contributing to the promotion of Eimeria reproduction.While only daily FI was observed in the current study, it would be interesting for future research to monitor daily BW changes to further support this hypothesis.The varying patterns in the birds' FI response to dietary Met levels on each DPI as Eimeria infection progressed could be attributed to certain physiological changes induced by the infection.Further investigation is necessary to elucidate the exact mechanism behind this change.
Nevertheless, for overall growth performance indices, increased Met levels linearly improved the BWG and FCR from 0 to 6 DPI.As the first limiting amino Table 10 Effects of dietary methionine levels and protein contents on liver oxidative status of broilers challenged with Eimeria spp.
a,b Means within a column lacking a common superscript differ (P < 0.05) 1 MDA, Malondialdehyde (μmol/L/mg of protein); SOD, superoxide dismutase (U/mg of protein); GPX, glutathione peroxidase (nmol/min/mg of protein); GSH, glutathione (μmol/L/mg of protein); GSSG, glutathione disulfide (μmol/L/mg of protein)  acid in the poultry diet [14], Met deficiency has been shown to hindered the growth performance of broilers raised under unchallenged conditions [21,57,58], this current research provided further evidence underscoring the crucial role of Met in sustaining the growth performance of the birds under Eimeria challenge.However, it is worth noting that although a linear trend between Met levels and performance indices were observed, this improvement in growth performance seemed to plateau when the Met levels reached 6.0 g/kg especially from 7 to 9 DPI where a quadratic trend was observed between FCR and Met levels.This observation suggested that the higher Met supplementation beyond this level might not necessarily lead to improvement in the growth performance for birds under Eimeria challenge.Interestingly, despite previous research showed that reduced CP levels in diets could compromise the performance of the birds [59,60], this decrease in growth performance caused by lower dietary CP was not observed in the current study.While the previous studies were conducted in unchallenged birds, the anorexia induced by coccidiosis and accompanying physiological changes in the current study might lead to a less prominent compromising effect of reduced CP levels on growth performance.This observation aligns with findings by Teng et al. [38], who also fed broilers with reduced CP diets under Eimeria challenge.
For the influence of Met levels and protein contents on intestinal morphology, we again observed that the protein contents affected the duodenal VH:CD ratios differently on two timepoints.On 6 DPI, the LCP groups had higher VH:CD than the NCP groups, while the opposite pattern was observed on 9 DPI.Similarly results have been reported by previous studies finding that reduced dietary protein resulted in higher duodenal VH:CD ratio in the acute infection phase while not in the recovery phase [38].In the acute infection phase, a reduction in dietary protein content could plausibly diminish the trypsin secretion, a factor responsible for the excystation of sporozoites from sporocysts [38,61,62].This attenuation may result in an elevated VH:CD ratio which was generally considered as an indicator for better morphology and absorption function [63][64][65].Conversely, during the recovery phase, the protein deficiency might have impeded the recovery processes, contributing to the lower VH:CD ratio in the LCP groups.A previous swine study has reported that the reduced protein diet negatively impacted the duodenal morphology [66].Regarding the quadratic trends observed in jejunal CD and duodenal VH:CD ratio with increasing Met levels in the current study, it suggested that both low and high Met levels might confer benefits to the intestinal morphology of broilers under coccidia challenge.Given the close association of Met with folate metabolism and the synthesis of SAM [37,67], and considering the significance of both compounds for parasite replication [25,39,40], lower Met levels could potentially lead to reduced Eimeria reproduction and lessened damage to the intestinal morphology by reducing the metabolism of folate and synthesis of SAM.On the other hand, the higher Met levels could mitigate the damage caused by infection due to the importance of Met in protein synthesis and its antioxidant capacity [14,15,68].Further research needs to confirm the above hypothesis and investigate the mechanism behind the observation.
The increased Met levels exhibited contrasting effects on the gene expression of tight junction proteins at both time points in the current study.On 6 DPI, increased Met levels linearly increased expression of CLDN1 and ZO2 in the jejunum, aligning with findings from prior studies where increased Met supplementation also elevated the tight junction protein expression in broilers [20,23].This underscores the importance of Met in preserving intestinal integrity; however, intriguingly, on 9 DPI, the linear increase in Met was associated with a decrease in the expression of these genes in the NCP groups.This provides additional evidence that birds underwent physiological changes during Eimeria infection, leading to the varied responses to dietary factors on different timepoints.As for the gene expression of amino acid transporters, lower Met levels were associated with higher expression of these genes, similar results have also been reported by Fagundes et al. [69] who fed the broilers diets with two different levels of Met.This observation suggested that the birds may have an alternative compensatory mechanism to address Met deficiency, distinct from merely increasing their FI.Increased Met levels quadratically affected the gene expression of cytokines in the NCP groups, a similar quadratic trend was reported in a previous research where the birds were fed diets containing graded levels of Met with normal CP content [21].However, such trend was not observed in the LCP groups, indicating a potential modulation in the regulatory pathways associated with cytokine gene expression and Met levels in the context of varying dietary protein contents.It is noteworthy that reducing the protein content led to a decrease in the gene expression of cytokines, underscoring the crucial role of protein in affecting certain aspects of immune responses of broilers as highlighted in serval previous review papers discussing the importance of protein or essential amino acids in regulating immune responses [11,[70][71][72].
The current study provided further evidence for the importance of Met in the production of GSH, the important endogenous antioxidants [26,73,74], as the GSH and GSSG contents linearly increased as Met level increased.This observation provided some support to the above proposed hypothesis that the higher Met level mitigated the damage of Eimeria infection to the intestinal morphology by alleviating the infectioninduced oxidative stress.While the essential role of Met in GSH synthesis has been documented in other studies [20,75,76], the results from the current study also suggested that the improvement of GSH or GSSG profiles by increasing Met levels seemed to be less prominent on 9 DPI when multiple previous studies have consistently demonstrated the oxidative stress caused by coccidiosis diminished as the birds recovered from the infection [4,20,21,77].These findings collectively underscored the essential relationship among Met, GSH production, and the birds' dynamic response to Eimeria infection over time.
The daily oocyst shedding trend corresponded to the parasite reproduction cycle, and the similar trend has been reported by previous research investigating the oocyst shedding in broilers infected with mixed species of Eimeria [78,79].The current study unveiled a linear or quadratic increase in oocyst shedding across all three species in response to elevated Met levels.This outcome substantiated the hypothesis that higher Met levels could promote the reproduction of Eimeria.A previous study has also reported that additional Met supplementation increased oocyst shedding in Eimeria-challenged broilers [38].While the lesion score was not assessed in the present study, since Met primarily impacts antioxidant capacity and immune regulation, observing marked differences in lesion scores may be less likely.
Nevertheless, in the future study, incorporating this assessment should be considered.To explore the potential mechanisms behind this increase in oocyst shedding, we examined whether the increase in Met level upregulated the expression of key enzymes associated with folate and SAM metabolism as previous studies have highlighted their intricate connection and essential roles in DNA synthesis and methylation for parasites [6,19,23,24].The results showed that the increased Met level only linearly increased the expression of TYMS in the LCP groups on 6 DPI, which is the key enzyme regulating the production of dTMP required for DNA synthesis and repair [80,81].More intriguingly, on 9 DPI the increased Met linearly or quadratically decreased the expression of these key enzymes.In other words, the birds increased the expression of these enzymes, possibly to compensate for the deficiency in dietary Met levels.Further studies may need to delve deeper into the analysis of the actual protein levels or activities of these enzymes to better understand how Met level may affect the metabolism of folate and SAM and in turn affect the reproduction of Eimeria.Additionally, the results showed that expression of these enzymes was lower in the LCP groups than the NCP groups, suggesting an existed interplay between protein content and metabolism of other nutrients.

Conclusion
In summary, in broilers challenged with Eimeria spp., although increasing Met levels improved the growth performance of the birds from 0 to 6 DPI, such improvement tended to plateau with the Met level reaching 6.0 g/kg.Interestingly, reducing the dietary CP did not deteriorate the growth performance of the birds.The dietary CP contents and Met levels exerted contrasting effects on intestinal morphology and tight junction protein expression on 6 and 9 DPI, whereas increasing Met consistently decreased the expression of amino acid transporters.Significant quadratic relationships were observed between Met levels and liver GSH concentrations, underscoring the role of Met in the antioxidant system.It was worth noting that the increasing Met levels led to increased oocyst shedding which might be related to the role of Met in folate metabolism.In conclusion, our results indicated that reducing the dietary CP level by 3% with 6.0 g/ kg of Met could maintain the performance and intestinal health of broilers under Eimeria challenge.Future study could seek to investigate the mechanism behind the increased oocyst shedding observed in this current study and its influences for subsequent flock performance on the same litter.
Table 13 Effects of dietary methionine levels and protein contents on expression of folate metabolism enzymes of broilers challenged with Eimeria spp.
a,b Means within a column lacking a common superscript differ (P < 0.05)

Fig. 4
Fig. 4 Effects of dietary methionine levels and protein contents on expression of tight junction proteins of broilers challenged with Eimeria spp.The error bars represent the SEM values.Bars without a common letter differ significantly.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, Day post inoculation; CLDN1, Claudin-1; ZO2, Zonula occludens 2; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Fig. 5
Fig. 5 Effects of dietary methionine levels and protein contents on expression of amino acid transporters of broilers challenged with Eimeria spp.The error bars represent the SEM values.Bars without a common letter differ significantly.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, day post inoculation; SLC7A9, b(0, +)-type amino acid transporter 1; SLC6A19, Sodium-dependent neutral amino acid transporter B(0)AT1; SLC43A2, L-type amino acid transporter 4; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Fig. 6
Fig. 6 Effects of dietary methionine levels and protein contents on expression of cytokines of broilers challenged with Eimeria spp.The error bars represent the SEM values.Bars without a common letter differ significantly.The asterisk (*) denotes significant differences.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, Day post inoculation; IL1β, Interleukin-1β; IL10, Interleukin-10; TNFα, Tumor necrosis factor α; TGFβ, Transforming growth factor β; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Fig. 7 Fig. 8
Fig. 7 Effects of dietary methionine levels and protein contents on liver oxidative status of broilers challenged with Eimeria spp. on 6 DPI.The error bars represent the SEM values.Bars without a common letter differ significantly.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, Day post inoculation; GSH, Glutathione; GSSH, Glutathione disulfide; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Fig. 9
Fig. 9 Effects of dietary methionine levels and protein contents on oocyst shedding of broilers challenged with Eimeria spp.The results were expressed as the log 10 of oocysts per gram of excreta.The error bars represent the SEM values.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.OPG, Oocyst per gram of excreta; DPI, Day post inoculation; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Fig. 10 Fig. 11
Fig. 10 Effects of dietary methionine levels and protein contents on expression of methionine metabolism enzymes of broilers challenged with Eimeria spp.The error bars represent the SEM values.Bars without a common letter differ significantly.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, Day post inoculation; MAT1A, Methionine adenosyltransferase 1A; MTR, Methionine synthase; CBS, Cystathionine beta synthase; AHCYL, Adenosylhomocysteinase like 1; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Table 3
Effects of dietary methionine levels and protein contents on post inoculation daily feed intake of broilers under Eimeria challenge Effects of dietary methionine levels and protein contents on the growth performance of broilers challenged with Eimeria spp.The error bars represent the SEM values.Bars without a common letter differ significantly.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, Day post inoculation; Fig.2

Table 4
Effects of dietary methionine levels and protein contents on growth performance of broilers under Eimeria challenge a,b Means within a column lacking a common superscript differ (P < 0.05)1DPI, Days post inoculation; BW, Body weight (g); BWG, Body weight gain (g); FI, Feed intake (g) 2 NCP, Normal protein group; LCP, Reduced protein group; Met, Methionine; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Table 5
Effects of dietary methionine levels and protein contents on intestinal morphology of broilers challenged with Eimeria spp. on 6 day post inoculation a,b Means within a column lacking a common superscript differ (P < 0.05) 1 VH, Villus height (μm); CD, Crypt depth (μm) 2 NCP, Normal protein group; LCP, Reduced protein group; Met, Methionine; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kgFig.3 Effects of dietary methionine levels and protein contents on intestinal morphology of broilers challenged with Eimeria spp.The error bars represent the SEM values.Bars without a common letter differ significantly.The asterisk (*) denotes significant differences.The black lines with arrowhead represented significant linear or quadratic relationship between parameters and dietary methionine levels.Statistical significance was set at P ≤ 0.05.DPI, Day post inoculation; CD, Crypt depth; VH, Villus height; Met, Methionine; NCP, Normal protein diet; LCP, Reduced protein diet; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg

Table 11
Effects of dietary methionine levels and protein contents on total oocyst shedding from 0 to 9 day post inoculation of broilers under Eimeria challenge a,b Means within a column lacking a common superscript differ (P < 0.05)1DPI, Days post inoculation; NCP, Normal protein group; LCP, Reduced protein group; Met, Methionine; Met 2.8, Dietary Met level = 2.8 g/kg; Met 4.4, Dietary Met level = 4.4 g/kg; Met 6.0, Dietary Met level = 6.0 g/kg; Met 7.6, Dietary Met level = 7.6 g/kg; Met 9.0, Dietary Met level = 9.0 g/kg2The results were expressed as the log 10 of oocysts per gram of excreta